Cultured Cell Sheet, Production Method and Tissue Repair Method Using Thereof

ABSTRACT

It is intended to provide a cultured cell sheet with excellent tissue adherence and flexibility. The above object can be achieved by culturing cells on a support for cell culture in which a surface of a substrate is coated with a temperature-responsive polymer whose lower or upper critical solution temperature against water is in the range of 0 and 80° C. along with a surfactant protein or a crosslinking inhibitor and producing a cultured cell sheet by detaching it by setting the temperature of the culture to the upper critical solution temperature or higher or to the lower critical solution temperature or lower.

TECHNICAL FIELD

The present invention relates to a cultured cell sheet, a productionmethod, and an applied tissue repair method thereof, in medical andbiological fields, etc.

BACKGROUND ART

Japan is becoming an aging society with the highest average lifeexpectancy in the world. People are beginning to place more emphasis onliving better, i.e., “the quality of life (QOL)”, rather than merelyprolonging life. Under such circumstances, medical techniques arerapidly progressing, and techniques for reconstruction of organs damagedby trauma or disease are also advancing remarkably. Therefore, in recentyears, a lot of attention has been focused on regenerative medicaltechniques in which organ tissue is reconstructed in the cell culturesystem employing cultured cells, and then transplanted to the targetsite.

To carry out such treatments, it is necessary to use tissue adhesiveagents for tissue adhesion. The tissue adhesive agents currently used inthe clinical field have been broadly classified into cyanoacrylateadhesive agents, gelatin-aldehyde adhesive agents, and fibrin glueadhesive agents. Cyanoacrylate adhesive agents employ adhesiveness by apolymerization reaction of the agents' cyanoacrylate monomers, and aresuperior in adhesive intensity and bonding speed. However, cyanoacrylateadhesive agents are synthetic agents which are not originallyendogenous, and prevent healing by producing formaldehyde byhydrolyzation of a cured monomer, which causes toxicity to livingorganisms. Therefore, the problems exist that the site to be applied waslimited, and the adhesive agents should not be applied to a site near acentral nerve or blood vessel. Gelatin-aldehyde adhesive agents employadhesiveness by a cross-linking reaction between a gelatin (biopolymerof the degenerated collagen) and formaldehyde or glutaraldehyde.However, the gelatin-aldehyde adhesive agents are also synthetic agentsthat are not endogenous. Although the gelatin-aldehyde adhesive agentsalso have sufficiently high adhesive intensity, since toxic aldehydecompounds are employed as a cross-linking agent, they are alsobiologically toxic. On the other hand, fibrin glue adhesive agents aremade from a tissue-derived material, which employs adhesiveness by areaction of blood coagulation. Although this type of adhesive agent isless toxic than the above described synthetic adhesive agents, theadhesiveness is low, and large amounts of fibrin glue should be used,since the fibrin glue itself is metabolized in vivo. Further, recently,some problems are being indicated relating to the topical inflammationat the site where the adhesive agent has been applied, since the fibringlue is prepared and purified heterogeneously or the mechanisms of theadhesiveness of the fibrin glue are the same as the blood coagulationreaction.

Technology that provides a cell sheet having sufficient basalmembrane-like proteins has been proposed. Conventionally, cell cultureis conducted on a glass surface, or on the surface of a syntheticpolymer compound along with a variety of surface processing. In order toachieve this, for example, various types of vessels made of polystyrenesubjected to surface processing such as silicone coating, gammairradiation, etc., are commonly used as vessels for cell culture. Cellsthat have been cultured and grown with these types of cell culturevessels, are detached and harvested from the surface of the vessel by achemical agent treatment or a proteinase treatment such as trypsin.However, in cases where the cells are harvested by the above-mentionedchemical agent treatment, some disadvantages have been pointed out: thetreatment method is cumbersome and complicated; the potential forcontamination by impurities is increasing; and examples of defects, inwhich cells are caused to degenerate or are damaged by the chemicaltreatment, and lose their original function.

Thus far, in order to overcome the above-mentioned disadvantages, anumber of techniques have been proposed by the present inventors.Especially, in Japanese Patent Application No. 2001-226141, a method forproducing a cultured cell sheet which comprises steps of coating thesurface of the cell culture support with a temperature responsivepolymer having a lower or upper critical solution temperature rangingfrom 0° C. to 80° C. in water, having the cultured cell layersmulti-layered by way of a conventional method, as necessary, anddetaching the cultured cell sheet only by changing the temperature ofthe culture support. As a result of application of this method, acultured cell sheet having sufficient strength can be produced.Furthermore, a thus obtained cultured cell sheet also retains basalmembrane-like proteins, and also has improved adhesiveness to tissue,when compared with a cell sheet harvested using the above describeddispase treatment. Moreover, PCT International Publication No. WO02/08387 discloses a method for producing a cultured myocardial cellsheet, which comprises steps of culturing the cells of myocardial tissueon a cell culture support having a support surface coated or coveredwith a temperature responsive polymer, preparing a myocardium-like cellsheet, and subsequently, adjusting a temperature of the culture mediumto a temperature greater than the upper critical solution temperature orless than the lower critical solution temperature, bringing the layeredcultured cell sheet into close contact with a polymer membrane,detaching the cultured intact cell sheet together with the polymermembrane, and three-dimensionally structuring by a predetermined method.As a result of application of this method, a myocardium-like cell sheetand a three-dimensional structure were discovered to be constructed invitro with reduced structural defects and with some of the functions ofmyocardial tissue. Neither of Application No. JP 2001-226141 nor WO02/08387 have investigated conferring the flexibility to the cell sheet,and have discussed the use as tissue repair material for suppressing theair leakage, blood leakage or bodily fluid leakage from the surface ofan organ. However, the use of such a cultured cell sheet as a tissueadhesive can be employed for the patient receiving tissue repair with anextremely high degree of safety, because the cell sheet can be preparedfrom the cell of the patient himself and, therefore, is highly safe.

DISCLOSURE OF THE INVENTION Problem(s) to be Solved

The present invention is intended to solve the above-mentioned problemsin conventional technology. Specifically, a purpose of the presentinvention is to provide a cultured cell sheet with excellent tissueadhesiveness and excellent flexibility. Moreover, a purpose of thepresent invention is to provide a method for manufacturing the culturedcell sheet and application as tissue repair material for suppressing theair leakage, blood leakage or bodily fluid leakage from the surface ofan organ.

Means for Solving the Problem(s)

The present inventors have conducted research and development, byinvestigating various aspects, in order to solve the above-mentionedproblems. As a result, it was discovered that a highly adhesive culturedcell sheet with excellent tissue adhesiveness and application as tissuerepair material for suppressing the air leakage, blood leakage or bodilyfluid leakage from the surface of an organ was obtained by the steps of:culturing cells, such as fibroblasts, cells of alveolar tissue, or cellsof myocardial tissue, on a cell culture support, the surface of which iscoated with a temperature responsive polymer; and afterwards, adjustinga temperature of the culture medium to a temperature greater than theupper critical solution temperature or less than the lower criticalsolution temperature; and detaching the cultured cell sheet. The presentinvention was completed based on the above-mentioned knowledge.

Specifically, the present invention provides a highly adhesive culturedcell sheet with a superior adhesiveness to the surface of a leaking siteon an organ and with an excellent flexibility.

The present invention provides a highly adhesive cultured cell sheet,which comprises a step of culturing at least one type of cell selectedfrom the group consisting of: fibroblasts, cells of alveolar tissue,cells of myocardial tissue, cells of hepatic tissue, cells of vasculartissue, mesenchymal stem cells, and adipose derived cells, on a cellculture support having a surface of the support coated with atemperature responsive polymer having an upper or lower criticalsolution temperature ranging from 0° C. to 80° C. in water, andthereafter, comprising the steps of: adjusting the temperature of theculture medium to a temperature greater than the upper critical solutiontemperature or less than the lower critical solution temperature;bringing the cultured cell sheet in close contact with a carrier; anddetaching the cultured cell sheet together with the carrier. The highlyadhesive cultured cell sheet obtained in the above-mentioned mannerdemonstrates superior adhesiveness to the surface of a leaking site onan organ, and therefore, cultured cell sheets having superioradhesiveness, such as those of the present invention, are sometimescalled, “highly adhesive cultured cell sheets”.

Moreover, the present invention provides a cultured cell sheet forapplication as tissue repair material for suppressing air leakage, bloodleakage or bodily fluid leakage from the surface of an organ.

In addition, the present invention provides a treatment method which isconducted by transplanting the highly adhesive cultured cell sheet tothe site of air leakage, blood leakage or bodily fluid leakage from thesurface of an organ.

EFFECT OF THE INVENTION

The highly adhesive cultured cell sheet obtained by the presentinvention has extremely high adhesiveness to a leaking site of thesurface of an organ and an excellent flexibility. Therefore, the use ofthe cell sheet of the present invention enables to suppress air leakage,blood leakage or bodily fluid leakage from the surface of an organ.Consequently, the present invention is extremely useful in biologicaland medical fields, etc., such as cell engineering and medicalengineering.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cultured cell sheet of alveolar cells 10 days after thestart of the culture, as indicated in Example 2;

FIG. 2 is a photograph showing the appearance of an air leakage model,as indicated in Example 2;

FIG. 3 is a photograph showing the appearance of the cultured cell sheetadhered to the site of air leakage, as indicated in Example 2;

FIG. 4 is a photograph showing the appearance of the site of air leakagewhich was closed by the cultured cell sheet, as indicated in Example 2;

FIG. 5 is a photograph showing the results of a hematoxylin-eosinstained tissue section from the site of air leakage which was closed bythe cultured cell sheet of the invention, as described in Example 2;

FIG. 6 is a photograph showing the results of an Azan stained tissuesection from the site of air leakage which was closed by the culturedcell sheet of the invention, as described in Example 2;

FIG. 7 is a photograph showing the appearance of a bleeding model, asindicated in Example 4;

FIG. 8 is a photograph showing the appearance of a site of bleedingwhich was closed by the cultured cell sheet, as indicated in Example 4;

FIG. 9 is a photograph showing the results of a hematoxylin-eosinstained tissue section from a site of bleeding of the liver which wasclosed by the cultured cell sheet, 4 weeks after transplantation, asindicated in Example 4;

FIG. 10 is a photograph showing the results of an Azan stained tissuesection from a site of bleeding in the liver which was closed by thecultured cell sheet of the invention, 4 weeks after transplantation, asindicated in Example 4;

FIG. 11 is a photograph showing the appearance of a bleeding model, asindicated in Example 5; and

FIG. 12 is a photograph showing the appearance of a site of bleedingwhich was closed by the cultured cell sheet, as indicated in Example 5.

PREFERRED MODE FOR CARRYING OUT THE INVENTION

The present invention provides a cultured cell sheet which has superioradhesiveness to the surface of an organ and has an excellentflexibility. Suitable cells for producing the cultured cell sheet of thepresent invention may include any one type or combination of two or moretypes of cells selected from the group consisting of: fibroblasts, cellsof alveolar tissue, cells of myocardial tissue, cells of hepatic tissue,cells of vascular tissue, mesenchymal stem cells, and adipose derivedcells. However, the present invention is not strictly limited to theabove-mentioned types of cells. In the present invention, the highlyadhesive cultured cell refers to a sheet which is prepared by the stepsof culturing each of the above-mentioned types of cells as a singlelayer on the surface of the culture support, and then, detaching thecell sheet from the support. Thus obtained cell sheet has a lower sidesurface which is contacted to the culture support at the time ofculture, and an upper side surface on the opposite side thereof. If thecells are cultured on the cell culture support, the surface of which iscoated or covered with a temperature responsive polymer having an upperor lower critical solution temperature ranging from 0° C. to 80° C. toin water indicated in the present invention, there will be an abundanceof adhesive proteins produced on the lower side surface of the cellsheet at the time the cells are cultured.

The cultured cell sheet of the present invention may or may not includea scaffold other than that produced by the cultured cells, such ascollagen, fibronectin, laminin, etc. However, it is not particularlylimited.

The cultured cell sheet of the present invention is constructed of atleast one type or a combination of two or more types of cells selectedfrom the group consisting of: fibroblasts, cells of alveolar tissue,cells of myocardial tissue, cells of hepatic tissue, cells of vasculartissue, mesenchymal stem cells, and adipose derived cells. These cellsare cells that are capable of expressing phenotype of the various typesof chondroid tissue mentioned above.

The highly adhesive cultured cell sheet of the present invention adheresextremely well to the leaking site of the surface of an organ. The highadhesiveness of the cultured cell sheet is achieved by maintaining itsflexibility and reducing the shrinkage of the cultured cell sheet thatis detached from the surface of the culture support.

The flexibility as demonstrated by the present invention is such that,after tissue has been covered by the cultured cell sheet, theabove-mentioned cultured cell sheet obstructs the performance of thetissue no more than 20%, preferably no more than 10%, and even morepreferably no more than 8%. The highly adhesive cultured cell sheet ofthe present invention adheres extremely well to the site of the leakageon the surface of the body tissue. In cases where the cultured cellsheet that is covering the tissue is rigid, the performance of thetissue is obstructed more than 20%, and the organ cannot exhibit theintact function thereof.

Regarding the suppression of the shrinkage of the cultured cell sheet, apercentage of shrinkage of the cultured cell sheet when detached fromthe surface of the culture support is preferably no more than 20%, morepreferably no more than 10%, and even more preferably no more than 5%,in either of the lengthwise directions of the sheet. If the percentageof shrinkage is no less than 20%, the detached cell sheet becomesflaccid, and unable to adhere to living tissue well, even when attachedto the living tissue. As a result, the detached cell sheet with no lessthan 20% shrinkage demonstrates the characteristics of the highlyadhesive cell sheet of the present invention.

A method for preventing the cultured cell sheet from shrinking is notparticularly limited in any way, and includes a method comprising thesteps of bringing a ring-shaped carrier with a center portion cutout inclose contact with the above-mentioned cell sheets at the time ofdetaching the cultured cell sheet from the culture support, anddetaching the cell sheet with the above-mentioned carrier.

The carrier that is used when detaching the highly adhesive culturedcell sheet has a structure for keeping the cell sheet of the presentinvention from shrinking, and a carrier such as that made of a polymermembrane, or a structure formed from a polymer membrane, or a metallicfixture, etc., may be used. For example, in cases where a polymer isused as the carrier material, polyvinylidene difluoride (PVDF),polypropylene, polyethylene, cellulose and derivatives thereof, papers,chitin, chitosan, urethane, etc., may be used as the specific materialof the carrier.

In the case of the present invention the term “close contact”hereinafter refers to having the cell sheet in a state in which it doesnot slip or slide on the carrier, in order to prevent the cell sheetfrom shrinking at the boundary between the cell sheet and carrier, andtherefore, it may be in close contact by being physically bonded, or itmay be in close contact via the fluid (for example, the culture medium,and other isotonic solutions) existing between each of the cell sheetand the carrier.

The shape of the carrier is not specifically limited in any way. Forexample, when transplanting the highly adhesive cultured cell sheetobtained, if a cut out portion of the carrier which is the same size orlarger than the transplantation site is utilized, greater convenienceresults since the cell sheet is only fixed to the portion surroundingthe cutout portion, and only a cell sheet with a cut-out portion thatfits the transplantation site can be transplanted.

The highly adhesive cultured cell sheet of the present invention may beprovided as a monolayered sheet, or as a layered sheet. Here, thelayered sheet can be prepared by layering sheets consisting solely ofthe highly adhesive cultured cell sheet or may be prepared by combiningthe highly adhesive cultured cell sheet with a sheet of other cells. Forexample, a cell sheet includes, but is not limited to, one which isprepared by layering the above-mentioned fibroblast cell sheet andanother fibroblast cell sheet, or a cell sheet that is prepared bylayering of a fibroblast cell sheet and a cell sheet derived from cellsother than those of the fibroblast cell sheet (for example, a cell sheetmade of cells of alveolar tissue), etc. In such cases, if at least twodifferent types of cells are employed, the different cellsintercellularly interact with each other, and therefore, a cell sheethaving the characteristic of even higher activity can be obtained.Furthermore, a position where the cell sheet is layered, the order ofthe layered cell sheets, and the number of the layered cell sheets arenot particularly limited in any way. However, depending on the coveredtissue, the structure of the layered sheet may be varied, by employing ahighly adhesive cell sheet on the uppermost layer, and so on.Furthermore, the number of the layered cell sheets is preferably no morethan 10, more preferably no more than 8, and even more preferably nomore than 4. The fibroblasts may also be viable even in an environmentin which basic nutrients are not fully supplied. However, a cell sheetconsisting of more than 10 layers of cell sheets is undesirable, sinceit is difficult to deliver oxygen and nutrients to the center portion ofthe layered cell sheets.

For example, the layered sheet of the present invention can be producedby employing a method, for example, but not limited to, the onedescribed below:

(1) a method of superimposing the highly adhesive cultured cell sheet inclose contact with the above-mentioned carrier by the steps of attachingthe first cell sheet in close contact with the carrier to the cellculture support, and afterwards, removing the carrier off the first cellsheet by the addition of a culture medium, and layering the second cellsheet by attaching the second cell sheet in close contact with acarrier, and repeating the steps;(2) a method of superimposing the highly adhesive cultured cell sheet inclose contact with the above-mentioned carrier by the steps of invertingthe first cell sheet in close contact with the carrier, fixing thecarrier side of the first cell sheet to the surface of the cell culturesupport, attaching a second cell sheet to the cell sheet side of thefirst cell sheet, removing the carrier off the first cell sheet byadding culture medium afterwards, and repeating the steps by attachinganother separate cell sheet;(3) a method in which two cell sheets each in close contact with acarrier are brought into close contact with each other at the cell sheetside; and(4) a method in which the cell sheet in close contact with the carrieris fitted to an affected area of the individual, and after the cellsheet is attached to living tissue, removing the carrier, and overlayinganother separate cell sheet on the affected area.

The highly adhesive cultured cell sheet of the present invention ischaracterized in that the basal membrane-like proteins between the celland support formed during the culture are not damaged by enzymes, suchas proteinases like dispase, trypsin, etc. Therefore, in order toproduce a cultured cell sheet with such a characteristic, the cellculture is preferably conducted on a surface of the cell culture supportcoated with a temperature responsive polymer.

The temperature responsive polymer that is used to coat the cell culturesupport has upper or lower critical solution temperature ranging from 0°C. to 80° C. in water, and more preferably a temperature ranging from20° C. to 50° C. in water. An upper or lower critical solutiontemperature that exceeds 80° C. is not preferable, since cells may die.Furthermore, an upper or lower critical solution temperature that islower than 0° C. is also not preferable, since it typically causes anextreme decrease in cellular growth rate or causes cell death.

The temperature responsive polymer to be used in the present inventionmay be either a homopolymer or a copolymer. Examples of such a polymermay include, for example, the polymer disclosed in Japanese PatentPublication No. H2-211865 (JP 2-211865 A). Specifically, for example,they may be obtained by polymerization or copolymerization of themonomer mentioned below. Monomers that can be used include, for example,(meth)acrylamide compound, N- (or N,N-di)alkyl substituted(meth)acrylamide derivative, or a vinyl ether derivative; in the case ofa copolymer, at least two of these monomers may be selected and used.Moreover, those monomers may be copolymerized with other monomers, orpolymers may be grafted together or copolymerized, or alternatively,mixtures of polymers and copolymers may be employed. If desired, thepolymers may be crosslinked to an extent that does not impair theirproperties.

The support that is to be covered with the temperature responsivepolymer may be chosen from among the glass, modified glass, compoundssuch as polystyrene and poly(methyl methacrylate), and all othersubstances that can generally be shaped, as exemplified by polymercompounds other than those compounds, and ceramics.

The method of covering the support with the temperature responsivepolymer is not limited in any particular way but one may follow themethods described in JP 2-211865 A. Specifically, the coating operationcan be achieved by either subjecting the support and the above-mentionedmonomers or polymers to electron beam (EB) exposure, γ-ray irradiation,ultraviolet irradiation, plasma treatment, corona treatment or organicpolymerization reaction or by means of physical adsorption as effectedby application of coating solutions or the kneading step.

The coating of the temperature responsive polymer is suitably in therange of 0.5 to 5.0 μg/cm², preferably 1.0 to 4.0 μg/cm², and morepreferably 1.2 to 3.5 μg/cm². If the coverage of the temperatureresponsive polymer is less than 0.5 μg/cm², the cells on the polymerwill not easily detach even if they are given a stimulus and theoperating efficiency is considerably lowered, which is not preferable.If, on the other hand, the coverage of the temperature responsivepolymer is greater than 5.0 μg/cm², cells will not easily adhere to thecovered area and adequate adhesion of the cells becomes difficult toachieve. The shape of the culture support of the present invention mayinclude, for example, but is not particularly limited to, a dish shape,multi-plate shape, flask shape, cell-insert shape, etc., may beemployed.

The composition of the culture medium for culturing the above-mentionedcells of the present invention is not particularly limited in any way,and any conventionally used may be employed at the time theabove-mentioned cells are cultured. In the cases that fibroblasts, cellsof alveolar tissue, cells of myocardial tissue, cells of hepatic tissue,cells of vascular tissue, mesenchymal stem cells, and adipose derivedcells are cultured, the culture medium may be prepared by, for example,supplementing an α-MEM culture medium, an F-12 culture medium, DMEMculture medium, or any mixture thereof, with 10% to 20% bovine serum, oroptionally with 50 μg/ml of ascorbic acid 2-phosphate in addition tobovine serum.

Moreover, the cultured cell sheet of the present invention is highlyflexible, and this flexibility is achieved by culturing cells underspecific culture conditions. However, the method thereof includes, forexample, but is not limited to, a method where a surfactant protein isadded to the culture medium, a method where alveolar cells which producethe surfactant protein are co-cultured, a method whereβ-aminopropylnitrile is added to the culture medium, or a method whereother types of collagen cross-linking inhibitor is added to the culturemedium. In cases where any one of the methods where a surfactantprotein, β-aminopropylnitrile, or other type of collagen cross-linkinginhibitor is added is used, the concentration thereof added to theculture medium is preferably at least 10 μM, more preferably at least100 μM, and even more preferably at least 200 μM. Moreover, since thecell sheet is not flexible when additive concentration is less than 10μM, and the shape of the cell sheet cannot be retained when the additiveconcentration is greater than 500 μM, neither of these additiveconcentrations is desirable.

The temperature of the culture medium is not particularly limited, aslong as the temperature is below the upper critical solutiontemperature, or above the lower critical solution temperature of thepolymer that is coated onto the surface of the support. However, itshould be appreciated that a low temperature, where cultured cells cannot proliferate, or high temperature region, where cultured cellsundergo cell death, is of course unsuitable. Culture conditions otherthan temperature are not particularly limited, and conventionalprocedures may be followed. For example, a culture medium used may be aculture medium to which a serum such as common fetal bovine serum (FCS)has been supplemented, or a serum-free culture medium, to which no serumhas been added.

When detaching and harvesting the cultured cells from the culturedsupport material by the method of the present invention, the highlyadhesive cultured cell sheet in close contact with the carrier can beremoved by the steps of bringing the cultured cell sheet in close withthe carrier, adjusting the temperature of the culture support attachedto the cells to a temperature above the upper critical solutiontemperature, or below the lower critical solution temperature of thepolymer coating the culture support, increasing the hydrophilicity ofpolymer coating the surface of the support, and detaching the culturedcell sheet in close contact with the carrier from the culture support,due to weakening of the attachment between the culture support and thecultured cell sheet. Moreover, the sheet may be detached either in theculture medium which is used to culture the cells, or in anotherisotonic solution, depending on the purpose.

In order to detach and harvest the highly adhesive cultured cell sheetat a high yield, a method in which the cell culture support is lightlytapped and shaken, or a method in which the culture medium is agitatedusing a pipet, etc., may be used alone, or in combination. In addition,when required, cultured cells may be washed with isotonic solution,prior to detaching and harvesting.

The highly adhesive cultured cell sheet of the present invention, whenharvested in the above-mentioned manner, are not damaged by proteinaseslike dispase, trypsin, etc., from the time of culture to the time ofdetachment of the cell sheet. Therefore, the highly adhesive cell sheetwhich has been detached from the support retains the intercellulardesmosome structure, has only a few structural defects, and exhibitshigh strength. Furthermore, the basal membrane-like proteins of thesheet of the present invention between the cell and support formed atthe time of culture are not damaged by enzymes, thus providing superioradhesion to affected area at the time of transplantation, and allowingimplementation of a very effective treatment to be possible. Morespecifically, in cases where conventional proteinases such as trypsinare used, the intercellular desmosomal structure and the basalmembrane-like proteins between the cell and support, etc., are notretained at all; and therefore, the cells are detached from the culturesupport in a individually separated condition. Although it is commonlyknown that, among proteinases, dispase can allow for detachment withabout 10% to 60% of the retained intercellular desmosomal structure, thebasal membrane-like proteins between the cell and support, etc., arealmost completely damaged, and the cell sheet obtained has only lowstrength. However, the cell sheet of present invention keeps no less 80%of the desmosomal structure and basal membrane-like proteins intact, andtherefore, provides the various effects described above.

The leakage site of the surface on the organ is not particularly limitedas long as it is a site at which air, blood, or bodily fluid is leakingfrom the organ surface. Examples of the leakage site of the surface onthe organ include the site of air leakage from lung tissue and the siteof bleeding from vascular tissue or liver tissue. The utilization of thehighly adhesive cultured cell sheet of the present invention for thesurface of the leakage site may include, for example, but is notparticularly limited to, a method in which the affected area is coveredwith the highly adhesive cultured cell sheet of the present invention.In such cases, the cultured cell sheet may be cut to appropriately fitthe size and shape of the affected area. In this way, the highlyadhesive cultured cell sheet of the present invention is able to adhereextremely well to the surface of leakage site on an organ surface, whichcannot be achieved by the prior art.

A method for fixing the highly adhesive cultured cell sheet to thesurface of an organ as demonstrated by the present invention is notparticularly limited in any way, and therefore, the cell sheet may besutured to living tissue, may be connected to living tissue with anadhesive agent capable of being used in vivo, or may only be attached tothe affected area without using either of these means, in order toquickly graft the highly adhesive cultured cell sheet to living tissueas shown by the present invention.

An application of the highly adhesive cultured cell sheet as shown inthe present invention is effective, for example, but not particularlylimited to, in treatments of the air leakage from lung tissue and thebleeding from vascular tissue or liver tissue.

In cases where the cultured cell sheet of the present invention is usedin transplantation to the surface of an organ, the cultured cell sheetis transplanted by removing the cell sheet off the carrier, afterfitting the cell sheet to the affected area. The method for removing thecell sheet off the carrier include, for example, but not particularlylimited to, a method of removing the cell sheet off the carrier bywetting the carrier in order to weaken the adhesiveness, or a method ofcutting the cultured cell sheet using a cutting tool such as a scalpel,forceps, a laserbeam, a plasma wave, etc. For example, in cases where acell sheet is in close contact with a carrier with a center portioncutout is employed, it is preferable to cut the cultured cell sheet tobe transplanted with a laserbeam, or the like, along the border of theaffected area, since it is possible to avoid attachment of the culturedcell sheet to an undesired area outside of the affected area.

The highly adhesive cultured cell sheet obtained by the method mentionedabove is superior when compared with one obtained by conventionalmethods, due to the noninvasiveness of the cultured cell sheet at thetime of detachment, and therefore, the clinical applications for theleakage site of the tissue for transplantation, etc., are verypromising. Especially, the highly adhesive cultured cell sheet of thepresent invention shows higher adhesiveness to living tissue than aconventional transplantation cell sheet, and grafts to living tissuevery rapidly.

Moreover, antigenic and infective problems can be solved due to the useof autologous cells. With respect to the fixation of the cultured cellsheet to the leakage site on the surface of an organ, since the culturedcell sheet of the present invention is non-invasively harvested togetherwith the extracellular matrix including the adhesion molecules secretedby the cultured cell sheet, which is transplanted, the cultured cellsheet has an advantage in early establishment of the leakage site on thesurface of an organ to be transplanted when it is transplanted to thesurface. Therefore, the present invention provides an extremelyeffective technology with improvements in the treatment efficiency ofthe affected area, and further alleviation of a burden on a patient.

EXAMPLES

Hereinafter, the present invention will be explained in further detailbased on the following Examples, which are not intended to limit thescope of the present invention in any way.

Examples 1 and 2

To a commercial culture dish with a diameter of 3.5 cm (Falcon 3001,manufactured by Becton Dickinson Labware), 0.07 ml of the solution ofN-isopropyl acrylamide monomer dissolved in isopropyl alcohol at aconcentration of 53% (Example 1) or 54% (Example 2) was applied. Theculture dish was exposed to electron beams at an intensity of 0.25 MGy,and the N-isopropyl acrylamide polymer (PIPAAm) was immobilized on asurface of the culture dish. After irradiation, the culture dish waswashed with ion-exchanged water to remove a residual monomer and thePIPAAm that did not bind to the culture dish, was then dried inside aclean bench, and sterilized by ethylene oxide gas, to obtain a cellculture support material coated with a temperature responsive polymer.

The amount of the temperature responsive polymer on the support surfacewas measured. As a result, it was found that the supports' surface wascoated with the temperature responsive polymer in an amount of 1.7μg/cm² (Example 1), and 1.9 μg/cm² (Example 2), respectively. A lungtissue was extracted from a GFP-transgenic neonatal rat, and the cellswere isolated with collagenase. Three days after the start of culture,the cells were subcultured on the above described cell culture supportmaterial, and the cell culture support was cooled for 30 minutes at 20°C. to harvest the cell sheet of the fourth passage. The cell sheet atthe time of harvest is shown in FIG. 1.

The harvested cell sheet was layered, and was applied to closing of airleakage. An air leakage model was prepared as follows: an 8 week oldF-344 nude rat was anesthetized intraperitoneally and placed on anartificial ventilator after endotracheal intubation; the left posteriorlateral side of the anesthetized rat was excised; the thorax was openedat the fourth intercostal space; the lung-pleura region was excisedapproximately 3 cm; and the air leakage was confirmed with a MinuteVolume of 400 cc. The air leakage site was then covered with adouble-layered cell sheet, the artificial ventilator was restarted after5 minutes of respiratory arrest, the Minute Volume was increased 100 ccevery 5 minutes to a maximum of 1000 cc, and the degree of adhesion ofthe cell sheet, and the presence or absence of air leakage wereevaluated.

The results showed that the above-mentioned cultured cell sheet wasattached to the air leakage site, was elongated and contracted inagreement with the artificial ventilator, and closed the air leakagesite. The appearance is shown in FIGS. 2 to 4 (FIG. 2 shows the airleakage model, FIG. 3 shows the cultured cell sheet attached to the airleakage site, and FIG. 4 shows air leakage being closed by the culturedcell sheet, respectively). Moreover, a histological evaluation wasconducted by a hematoxylin-eosin staining or an Azan staining of tissuesections of the tissue with the closed air leakage site. The resultsobtained are shown in FIG. 5 (hematoxylin-eosin stain), and FIG. 6 (Azanstain).

From both figures, it was clear that the cultured cell sheet used forcovering the leakage site was in close contact with the surface of thetissue around the air leakage site. Therefore, the usefulness of thecultured cell sheet as a tissue repair material could be confirmed.

Example 3

The study of this example is conducted in a similar manner to that ofExample 2, except that 250 μM β-aminopropylnitrile was added to theculture medium, when cell passage was started on the cell culturesupport material, 3 days after the start of culture in Example 2. Thecultured cell sheet obtained in the presence of β-aminopropylnitrile wasmechanically flexible. The air leakage site was closed, and contractureof the covered portion of the cultured cell sheet was not found.Accordingly, the usefulness of a cultured cell sheet having flexibilityas a tissue repair material could be confirmed.

Example 4

A lung tissue was extracted from a GFP-transgenic neonatal rat, and thecells were isolated with collagenase, in a manner similar to that ofExample 2. Three days after the start of culture, the cells weresubcultured on the above described cell culture support material, andthe cell culture support was cooled for 30 minutes at 20° C. to harvestthe cell sheet of the fourth passage.

The harvested cell sheet was layered, and was applied to closing thebleeding site of the liver. A model for a bleeding site of the liver wasprepared as follows: an 8 week old F-344 nude rat was anesthetizedintraperitoneally and placed on an artificial ventilator afterendotracheal intubation; the abdominal area of the anesthetized rat wasexcised; the surface layer of the liver was excised approximately 2 mm;and bleeding was confirmed. The site of bleeding was then covered with adouble-layered cultured cell sheet, and the degree of adhesion of thecell sheet, and the presence or absence of bleeding were evaluated.

The results showed that the above-mentioned cultured cell sheet wasattached to the bleeding site, and that the bleeding site was closed.The appearance is shown in FIGS. 7 and 8 (FIG. 7 shows the bleedingmodel, and FIG. 8 shows the cultured cell sheet attached to the bleedingsite, respectively). Moreover, a histological evaluation was conductedby a hematoxylin-eosin staining or an Azan staining of tissue sectionsof the tissue with the closed bleeding site of the liver four weeksafter transplantation for closing the bleeding site. The resultsobtained are shown in FIG. 9 (hematoxylin-eosin stain), and FIG. 10(Azan stain).

From both figures, it was clear that the cultured cell sheet used forcovering the bleeding site was in close contact with the surface of thetissue around the bleeding site. Therefore, the usefulness of thecultured cell sheet as a tissue repair material could be confirmed.

Example 5

A lung tissue was extracted from a GFP-transgenic neonatal rat, and thecells were isolated with collagenase, in a manner similar to that ofExample 1. Three days after the start of culture, the cells weresubcultured on the above-described cell culture support material, andthe cell culture support was cooled for 30 minutes at 20° C. to harvestthe cell sheet of the fourth passage.

The harvested cell sheet was layered, and was applied to closing thebleeding site of the blood vessel. A model for a bleeding site of ablood vessel was prepared as follows; an 8 week old F-344 nude rat wasanesthetized intraperitoneally and placed on an artificial ventilatorafter endotracheal intubation; the abdominal area of the anesthetizedrat was excised; a blood vessel was excised with a suture needle; andbleeding was confirmed. The site of bleeding was then covered with adouble-layered cultured cell sheet, and the degree of adhesion of thecell sheet, and the presence or absence of bleeding were evaluated.

The results showed that the above-mentioned cultured cell sheet wasattached to the bleeding site of the blood vessel, and that the bleedingfrom the site was stopped. The appearance is shown in FIGS. 11 and 12(FIG. 11 shows the bleeding model, and FIG. 12 shows the cultured cellsheet attached to the bleeding site, respectively).

From both figures, it was clear that the cultured cell sheet used forcovering the bleeding site was in close contact with the surface of thetissue around the bleeding site. Therefore, the usefulness of thecultured cell sheet as a tissue repair material could be confirmed.

INDUSTRIAL APPLICABILITY

The highly adhesive cultured cell sheet obtained by the presentinvention has extremely high adhesiveness to the leakage site of thesurface the surface of an organ, and an excellent flexibility. The useof the cell sheet of the present invention enables suppression of airleakage, blood leakage or bodily fluid leakage from the surface of anorgan. Therefore, the present invention is very promising in itsclinical applications to air leakage from alveolar tissue or bleedingfrom blood vessel tissue or liver tissue, etc. Consequently, the presentinvention is extremely useful in biological and medical fields, etc.,such as cell engineering and medical engineering.

1. A cultured cell sheet, having superior tissue adhesiveness andexcellent flexibility.
 2. The cultured cell sheet according to claim 1,wherein the cells are selected from the group consisting of:fibroblasts, cells of alveolar tissue, cells of myocardial tissue, cellsof hepatic tissue, cells of vascular tissue, mesenchymal stem cells, andadipose derived cells.
 3. The cultured cell sheet according claim 1 or2, wherein the cultured cell sheet is a layered cultured cell sheet. 4.The cultured cell sheet according to claim 3, wherein the layered cellsheet is prepared by layering of the cell sheet of claim
 2. 5. Thecultured cell sheet according to any one of claims 1 to 4, wherein thesuperior adhesiveness allows the cultured cell sheet to cover a site ofair leakage, blood leakage or bodily fluid leakage on tissue, andthereby to suppress such leakage.
 6. The cultured cell sheet accordingto any one of claims 1 to 5, wherein the cultured cell sheet has aflexibility such that the cultured cell sheet obstructs performance ofthe tissue not more than 20% after tissue has been covered by thecultured cell sheet.
 7. The cultured cell sheet according to any one ofclaims 1 to 6, wherein the flexibility results from a surfactantprotein.
 8. The cultured cell sheet according to claim 7, wherein thesurfactant protein is produced by the cultured cell sheet.
 9. Thecultured cell sheet according to any one of claims 1 to 8, wherein theflexibility results from a cross-linking inhibitor.
 10. The culturedcell sheet according to claim 9, wherein a cross-linking inhibitor is acollagen cross-linking inhibitor, β-aminopropylnitrile.
 11. The culturedcell sheet according to any one of claims 1 to 10, wherein the culturedcell sheet is used for a treatment for suppressing air leakage, bloodleakage or bodily fluid leakage from a surface layer of an organ. 12.The cultured cell sheet according to claim 11, wherein the treatmentcomprises covering the surface of an affected portion of an organ withthe cultured cell sheet.
 13. The cultured cell sheet according to eitherclaim 11 or 12, wherein the subject to be treated is air leakage fromlung tissue, bleeding from liver tissue or bleeding from vasculartissue.
 14. The cultured cell sheet according to any one of claims 11 to13, wherein the cultured cell sheet is cut according to the size andshape of the affected area, before covering of the surface of theaffected area.
 15. A method for preparing a cultured cell sheet, whereincells are cultured on a cell culture support having a surface coatedwith a temperature responsive polymer having an upper or lower criticalsolution temperature of 0° C. to 80° C. in water, and, thereafter,comprising steps of: (1) adjusting the temperature of the culture mediumto a temperature above the upper critical solution temperature or belowthe lower critical solution temperature; and (2) detaching the culturedcell sheet.
 16. The method according to claim 15, wherein thetemperature responsive polymer is poly(N-isopropyl acrylamide).
 17. Themethod according to either claim 15 or 16, wherein the cell sheet is nottreated with a proteolytic enzyme upon being detached.
 18. The methodaccording to any one of claims 15 to 17, wherein β-aminopropylnitrile isadded to the culture medium.
 19. A treatment method comprisingtransplanting the cultured cell sheet of any one of claims 1 to 14 tothe affected area of the surface layer of the organ from which air,blood or bodily fluid is leaking.
 20. The method according to claim 19,wherein the transplantation is conducted by covering of the surfacelayer of the affected area of the organ.
 21. The method according toclaim 19 or 20, wherein the cultured cell sheet is cut according to thesize and shape of the affected portion, before covering of the surfacelayer of the affected area of the organ.
 22. The method according to anyone of claims 19 or 21, wherein the subject to be treated is air leakagefrom alveolar tissue, bleeding from liver tissue, or bleeding fromvascular tissue.